Pectin polymers are important constituents of plant primary cell walls. They are composed of chains of 1,4-linked .alpha.-D-galacturonic acid and methylated derivatives thereof. The use of pectin-degrading enzymes such as polygalacturonase, pectin methylesterase, pectin lyase or pectate lyase is important for the food industry, primarily in fruit and vegetable processing such as fruit juice production or wine making, where their ability to catalyse the degradation of the backbone of the pectin polymer is utilised.
For many purposes, it would be desirable to provide each of the pectin degrading enzymes present in, for instance, commercial preparations containing a number of different pectin degrading enzymes (an example of such a preparation is Pectinex Ultra SP.RTM., prepared from Aspergillus aculeatus, available from Novo Nordisk A/S) in a form free from other components. In this way, it would be possible to produce enzyme preparations adapted to specific purposes, such preparations either containing a single pectin degrading enzyme or arbitrary combinations thereof. To serve this end, it is convenient to provide single-component pectin degrading enzymes by recombinant DNA techniques.
Pectin methylesterase (EC 3.1.1.11) catalyses the removal of methanol from pectin, resulting in the formation of pectic acid (polygalacturonic acid).
Pectin methylesterases are produced by a number of microorganisms and have also been found to be present in leaves, roots, stalks and fruits of many higher plants. Microbial pectin methylesterases have been cloned by Khanh et al. (1990) "Nucleotide and derived amino acid sequence of a pectinesterase cDNA isolated from Aspergillus niger strain RH5344", Nucleic Acids Res. 18:4262; Khanh et al. (1991) "Characterization and expression of a genomic pectin methyl esterase-encoding gene in Aspergillus niger", Gene 106:71-77; Spok et al. (1991) "Molecular cloning and sequencing of a pectinesterase gene from Pseudomonas solanacearum", J. Gen. Microbiol. 137:131-140; Plastow (1988) "Molecular cloning and nucleotide sequence of the pectin methyl esterase gene of Erwinia chrysanthemi B374", Mol. Microbiol. 2:247-254; Laurent et al. (1993) "Characterization and overexpression of the pem gene encoding pectin methylesterase of Erwinia chrysanthemi strain 3937", Gene 131:17-25; Tierny et al. (1994) "Molecular cloning and expression in Escherichia coli of genes encoding pectate lyase and pectin methylesterase activities from Bacteroides thetaiotaomicron", J. Appl. Bacteriol. 76:592-602.
WO 94/25575 describes the cloning of a pectin methylesterase from Aspergillus aculeatus.